Abstract The NF-?B essential modulator (NEMO) is an essential component in the activation of the canonical NF-?B pathway and exerts its function by recruiting the inhibitor of ?B kinases IKK? and IKK? to the IKK complex. Inhibition of the interaction between the IKK? and IKK? kinases and NEMO is considered an attractive therapeutic avenue to the inhibition of NF-?B, due to the pathway?s role in human diseases, encompassing inflammatory and autoimmune diseases and cancer. The strategy was shown to be effective utilizing a peptide, corresponding to the NEMO-binding domain of the IKKs (NBD), as an IKK complex inhibitor in in vitro and in vivo models of disease. The overall goal of the project is the understanding of the mechanism for NEMO inhibition and its exploitation for the development of small molecule inhibitors. The long- term expectation of the project is to establish the structural basis for inhibitor design and the requirements for high affinity binding and specificity. The project will evolve through three specific aims: 1. Structural and computational methods will be utilized to characterize the structure of NEMO-inhibitor complexes, which rely on a new construct of the IKK-binding domain of NEMO which easily yields X-ray structures. Furthermore, the available structures of unbound NEMO in a closed and semi-open structure will be utilized for inhibitor design. 2. The NEMO structures will guide the rational design of peptide and small molecule inhibitors, to develop into potent inhibitors with distinct physical and chemical properties from the NBD and testing the potential of different binding sites for inhibition. 3. The interaction between NEMO and inhibitors will be characterized by biochemical, biophysical and cellular methods, to quantify the binding affinity, validate binding, identify the binding site and characterize the binding mode and requirements. In addition, the biological evaluation in cellular model systems of cancer that rely on NF-?B signaling for survival, will provide the rationale and justification for future preclinical development and clinical studies. The expertise of the Investigators involved in the research matches the multidisciplinary character of the project, with experience in assay development and biophysical characterization, NMR-based screening and structure determination, X-ray crystallography, peptide chemistry and synthetic medicinal chemistry, cancer biology and anticancer drugs, and a long experience in the characterization of NEMO. The outcome of this research will further our understanding of the requirements and consequences of NEMO inhibition and open the way to the development of new therapies for human disease.